Community Sequencing Program: Project Proposal Proposer’s Name: David HIBBETT, Dan CULLEN, Dan EASTWOOD, Francis MARTIN, Antonio PISABARRO, and Igor GRIGORIEV Project Title: Community proposal to sequence a diverse assemblage of saprotrophic Basidiomycota (Agaricomycotina) Proposal ID: A) Brief description: Abstract: We propose a community-based sequencing project for whole-genome sequencing of a suite of saprotrophic (decayer) Fungi in the Basidiomycota, subphylum Agaricomycotina. The proposed organisms are of central relevance to the DOE mission with regard to lignocellulose bioconversion, biofuel production, feedstock improvement, and carbon cycle functioning. This proposal is derived from the basidiomycete focus group of the JGI Fungal Genomics Program, which commenced in October 2009. The organisms targeted in this proposal are particularly relevant to the “Biorefinery” section of the Genomic Encyclopedia of Fungi that the FGP seeks to create. Scope of Work: We propose a suite of 30 species for genome sequencing, divided into two Tiers of 13 and 17 species each. This set of taxa includes all of the 24 saprotrophic species identified by the Basidiomycota community in response to the 2009 FGP. The Tier 1 taxa are already being processed as a “pilot project” of the FGP. We therefore propose the remaining seventeen species in Tier 2 for sequencing as a follow-up to the current pilot project. The taxa have been selected on the basis of five major criteria: 1) phylogenetic diversity, 2) functional diversity and ecological importance, 3) utility as experimental systems, and community interest, 4) complementarity and relevance to other CSP proposals, and 5) availability of starting materials (monokaryons) and experimental tractability. The chosen taxa represent fourteen of the approximately eighteen major clades (orders and subclasses) of Agaricomycotina, including five independent brown rot lineages. This selection has been developed in coordination with a parallel proposal by Francis Martin and colleagues to sequence the genomes of twenty-three ectomycorrhizal (ECM) taxa, including 18 Agaricomycotina and 5 Ascomycota. Together, the genomes of the saprotrophic taxa proposed here and the ECM taxa being proposed by Martin et al. will provide insight into the functional diversity of Agaricomycotina, including the genetic bases of transitions between saprotrophic and ECM lifestyles, as well as between white rot and brown rot. A third parallel proposal by Joseph Spatafora and colleagues proposes to sequence an assemblage of diverse Fungi representing undersampled branches of the fungal Tree of Life, with an emphasis on basal fungal lineages (the paraphyletic “chytrids” and “zygomycetes”). Several Basidiomycota are targeted in the Tree of Life proposal, but none are in the Agaricomycotina. The primary goals of the proposed genome sequencing effort are to describe gene content and facilitate functional (expression) and evolutionary analyses. It is understood that the sequencing technology is rapidly evolving. We therefore defer decisions regarding the optimal approach to WGS of these haploid monokaryons to the JGI staff. Illumina-based transcriptome analysis, specifically focused on colonized wood, is also being requested in this proposal. Augmenting the genome and transcriptome data, the PIs and collaborators will perform mass spectroscopy-based secretome analysis, wood compositional analysis and microscopy, all under standardized conditions. Finally, we will perform comparative phylogenetic analyses to assess the evolution of decay mechanisms in Agaricomycotina. B) Background information (Limit 3 pages) Technical Information: Direct information about genome size, G+C content, polymorphism level, and repeat structure is not available for the target taxa. Genomes for Agaricomycotina range from 19 Mbp in Cryptococcus neoformans (a pathogenic yeast in the Tremellomycetes) to 65 Mbp in Laccaria bicolor (an ectomycorrhizal mushroom in the Agaricales). The genome sizes for wood decay fungi have ranged from 35 Mbp (Phanerochaete chrysosporium, Polyporales) to 47 Mbp (Serpula lacrymans, Boletales). Based on comparisons with these fungi, the species proposed here are unlikely to have a problematic G+C content and the haploid genome sizes probably range from 30 to 50 Mb. Similarly, substantial amounts of repetitive DNA in the form of class I and II transposons and their remnants are expected. Up to 10% of the genome might be repetitive, although this is no longer particularly daunting for the JGI assembly team. (Another basidiomycete successfully sequenced by the JGI, Laccaria bicolor (Martin et al. 2008), was found to contain over 20% repeats and further complicated by a substantial amount of endobacterial DNA ‘contamination’.) Polymorphisms related to ploidy, which were problematic in the JGI Postia placenta genome project (Martinez et al. 2009), will be circumvented by sequencing monokaryons exclusively. Available Resources: Most of the taxa targeted here have not been used as model experimental systems, so there is little preliminary information about genome structure (e.g., physical maps, genetic maps, fingerprinted BAC libraries, etc). However, seven species (Bjerkandera adusta, Dichomitus squalens, Coniophora puteana, Fomitiporia mediterranea, Gloeophyllum trabeum, Phlebia brevispora, Trametes versicolor) have been used in various studies of decay chemistry, and there is published information on decay capabilities (e.g., ability to degrade crystalline cellulose by Gloeophyllum and Coniophora) and selected enzyme families (e.g., class II peroxidases and laccases in Bjerkandera adusta, Fomitiporia mediterranea, Phlebia brevispora, and Trametes versicolor) (Baldrian and Valaskova 2008, Lundell et al. 2010, Morgenstern et al. 2008). Genetic transformations systems are available for T. versicolor. Additionally, two species proposed here, Phanerochaete velutina and Phanerochaete flavido-alba, are closely related to the model white rot species Phanerochate chrysosporium, for which a complete genome sequence is available (Martinez et al. 2004). Technical Challenges: One of the Tier 2 target species, Hygrocybe sp., has yet to be successfully cultured on standard mycological media (e.g., malt-extract agar, potato-dextrose agar). We will attempt to obtain this organism as a monokaryotic culture using various enhanced media, such as Melins-Norkrans medium, vitamin-enhanced media, etc. Another Tier 2 target species, Sphaerobolus stellatus, is culturable, but attempts to obtain monokaryons have failed (it is possible that this species produces dikaryotic spores). We have identified several alternative taxa that are known to be culturable; Hydnomerulius pinastri or Marasmius spp. are alternatives for Hygrocybe sp., and Lentaria michneri is an alternative for Sphaerobolus stellatus. We will pursue monokaryotic cultures of the alternative taxa at the same time that we attempt to obtain monokaryotic cultures of Hygrocybe sp. and Sphaerobolus stellatus. Starting Materials: Saprotrophic Agaricomycotina are relatively easy to culture in both dikaryotic and monokaryotic forms and excellent culture collections exist in the United States and abroad. As noted, nucleic acids of thirteen monokaryotic strains have already been submitted and are in various stages of genome sequencing as a pilot project. Monokaryotic cultures for eight of the remaining 17 species have been located in the culture collections of the USDA Forest Products Laboratory (FPL), the University of Tennessee (TENN; laboratory of Ronald H. Petersen), and the Belgian Coordinated Collections of Microorganisms/Mycothèque de l'Universite catholique de Louvain (BCCM/MUCL; one species). Monokaryons of Phanerochaete velutina and Phanerochaete flavido-alba have not been located, but monokaryons of other closely related Phanerochaete species are present in FPL and could be substituted if necessary. Monokaryons of seven species will need to be located elsewhere or obtained from fruiting bodies collected from nature. Isolates in culture collections are occasionally misidentified. We will confirm identities of all cultures using sequences of the internal transcribed spacers (ITS1-2) of nuclear ribosomal genes. The PIs of the present proposal will obtain monokaryotic cultures of all of the target taxa from established culture collections and from new collections in nature, beginning in the summer of 2010. We anticipate that new DNA and RNA preparations will begin to be available for submission to JGI by the end of 2010. To aid annotation, we will continue to provide total RNA from various complex and defined media. For quantitative transcriptome analysis, total RNA and mRNA is isolated from wood using a system devised in Bob Blanchette’s laboratory (letter attached) (Vanden Wymelenberg et al. 2006). This method substantially reduces sample variation and involves thin wood wafers placed directly on actively growing mycelia. Wood wafers (1 cm X 1 cm X 2 mm) are cut from freshly harvested sapwood, sterilized and inoculated by contact with mycelium growing on malt extract agar (1.5% Difco malt extract and 1.5% agar liter-1) in Petri plates. Up to 100 ug high quality total RNA can be purified by simple modifications of the Qiagen RNEasy system. If Illumina transcriptome analysis is approved, mRNA may be preferred. If so, we can easily and efficiently purify mRNA from the colonized wafers by magnetic capture techniques. The wood-derived mRNA is an excellent template for cDNA synthesis. Ultimately, colonized